Kazuhiro Shibata

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis . Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Full-length complementary DNAs (cDNAs) are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We isolated 155,144 RIKEN Arabidopsis full-length (RAFL) cDNA clones. The 3′-end expressed sequence tags (ESTs) of 155,144 RAFL cDNAs were clustered into 14,668 nonredundant cDNA groups, about 60% of predicted genes. We also obtained 5′ ESTs from 14,034 nonredundant cDNA groups and constructed a promoter database. The sequence database of the RAFL cDNAs is useful for promoter analysis and correct annotation of predicted transcription units and gene products. Furthermore, the full-length cDNAs are useful resources for analyses of the expression profiles, functions, and structures of plant proteins.

We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 × 107/10 μg starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.

In the effort to prepare the mouse full-length cDNA encyclopedia, we previously developed several techniques to prepare and select full-length cDNAs. To increase the number of different cDNAs, we introduce here a strategy to prepare normalized and subtracted cDNA libraries in a single step. The method is based on hybridization of the first-strand, full-length cDNA with several RNA drivers, including starting mRNA as the normalizing driver and run-off transcripts from minilibraries containing highly expressed genes, rearrayed clones, and previously sequenced cDNAs as subtracting drivers. Our method keeps the proportion of full-length cDNAs in the subtracted/normalized library high. Moreover, our method dramatically enhances the discovery of new genes as compared to results obtained by using standard, full-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

We have developed a new class of cloning vectors: lambda-full-length cDNA (lambda-FLC) cloning vectors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, lambda-FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain "size balancers." Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre-lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors.

Many genes are known to be involved in gonadal differentiation in vertebrates. Dmrt1, a gene that encodes a transcription factor with a DM-domain, is considered to be one of the essential genes controlling testicular differentiation in mammals, birds, reptiles, amphibians and fish. However, it still remains unknown which testicular cells of animals other than mice and chicks express Dmrt1 protein. For an explanation of its role(s) in testicular differentiation in vertebrates, the expression of the Dmrt1 protein needs to be studied. For this purpose, we conducted an immunohistochemical study of this protein in an amphibian by using an antibody specific for Dmrt1. No positive signal was found in the indifferent gonad of tadpoles of Rana rugosa at early stages. However, in the testis of tadpoles at later stages (XV-XXV) and in frogs one month after metamorphosis, this protein was expressed in interstitial cells and Sertoli cells. In the testis of adult frogs, germ cells also expressed Dmrt1 protein. RT-PCR analysis revealed that the gene for this protein was not transcribed at any time during ovarian development, but was expressed in the female to male sex-reversed gonad. This was true when immunohistological studies were performed. In addition, Southern blot analysis showed DMRT1 to be an autosomal gene. Taken together, our findings indicate that Dmrt1 protein is expressed by interstitial cells, Seroli cells and germ cells in the testis of R. rugosa. Dmrt1 may thus be very involved in the testicular differentiation of amphibians.

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3' end and 5' end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month.

To enhance the usefulness of the laboratory mouse and to facilitate the rapid assay of gene functions we have been collecting the entire set of mouse full-length cDNA by one-pass sequencing. To collect full-length cDNA clones efficiently, it is critical to construct high-quality cDNA libraries. In recent years, we have been developing a way to construct full-length cDNA libraries by using biotinylation of the cap structure (the 'CAP-trapper' method) coupled with treatment to increase reverse transcriptase efficiency at high temperature by the addition of trehalose. In this paper we report our evaluation of the quality of CAP trapper and a number of other full-length cDNA libraries, including the results of 5' end analysis of clones in CAP trapper and the other libraries. We used a procedure that compared the 5'-ends of cDNA clones with those of genes in the public databases. Our analysis showed that 63% of cDNA clones in CAP trapper libraries had sequences that were either the same length as those of equivalent genes in the public database or 5'-extended, and that 90% of these clones maintained their coding sequences. These results indicate that the CAP trapper library is a promising tool for collecting full-length cDNA in large-scale projects. Comparison of the quality of CAP trapper with that of other full-length-cDNA libraries confirmed the value of these libraries.

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes.

We describe a computer-based method that selects representative clones for full-length sequencing in a full-length cDNA project. Our method classifies end sequences using two kinds of criteria, grouping, and clustering. Grouping places together variant cDNAs, family genes, and cDNAs with sequencing errors. Clustering separates those cDNA clones into distinct clusters. The full-length sequences of the clones selected by grouping are determined preferentially, and then the sequences selected by clustering are determined. Grouping reduced the number of rice cDNA clones for full-length sequencing to 21% and mouse cDNA clones to 25%. Rice full-length sequences selected by grouping showed a 1.07-fold redundancy. Mouse full-length sequences showed a 1.04-fold redundancy, which can be reduced by approximately 30% from the selection using our previous method. To estimate the coverage of unique genes, we used FANTOM (Functional Annotation of RIKEN Mouse cDNA Clones) clusters (). Grouping covered almost all unique genes (93% of FANTOM clusters), and clustering covered all genes. Therefore, our method is useful for the selection of appropriate representative clones for full-length sequencing, thereby greatly reducing the cost, labor, and time necessary for this process.

Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected into the wells from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hr. Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing.

Abstract This study reconsiders Francis Bacon’s ideas on spirits, death, and the prolongation of life through a chronological examination of his works. His conception of death has often been considered unique because it presupposed a common material basis for the dissolution of inanimate things and the death of human beings. However, his focus on this commonality seems to have faded gradually – though not completely – as his works progressed, from De viis mortis to his later works, including Historia vitae et mortis . He became increasingly conscious of the difference between the inanimate and the animate. While De viis mortis insisted on the role of inanimate spirits in aging and death, Historia vitae et mortis tended to consider vital spirits as the chief cause of human death. My suggestion in this article is that as Bacon’s ideas developed, they came closer to traditional conceptions of aging and death.

We developed computer-based methods for constructing a nonredundant mouse full-length cDNA library. Our cDNA library construction process comprises assessment of library quality, sequencing the 3′ ends of inserts and clustering, and completing a re-array to generate a nonredundant library from a redundant one. After the cDNA libraries are generated, we sequence the 5′ ends of the inserts to check the quality of the library; then we determine the sequencing priority of each library. Selected libraries undergo large-scale sequencing of the 3′ ends of the inserts and clustering of the tag sequences. After clustering, the nonredundant library is constructed from the original libraries, which have redundant clones. All libraries, plates, clones, sequences, and clusters are uniquely identified, and all information is saved in the database according to this identifier. At press time, our system has been in place for the past two years; we have clustered 939,725 3′ end sequences into 127,385 groups from 227 cDNA libraries/sublibraries (see http://genome.gse.riken.go.jp/ ). [The sequence data described in this paper have been submitted to the DDBJ data library under accession nos. AV00011–AV175734, AV204013 – AV382295 , and BB561685 – BB609425 .]

We have developed an RLGS-based scanning system to detect DNA alteration in tumor tissues, using 575 mapped spots/loci in a single gel. This system is very powerful for screening and identifying not only loss of heterozygosity (LOH) but also DNA methylation change. In this study, we applied this system to search for the LOH of hepatoma from an interspecific F1 hybrid between Mus spretus and C57BL/6 with SV40 early T antigen transgene connected to a mouse major urinary protein enhancer/promoter. Comparing the RLGS profiles of each tumor to that of the normal tissue showed significant LOH in chromosomes 1, 5, 7 and 13.

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed.

DNA methylation is the only known mechanism for an epigenetic genomic DNA modification that is capable of altering gene expression. A recent study reveals that the pattern of CpG island methylation is largely characteristic of tumor type, suggesting that distinct sets of genes are inactivated by methylation during development of each tumor type. We compared previously the methylation status between normal liver and liver tumors in SV40 T/t antigen transgenic mice (MT-D2 mice) using Restriction Landmark Genomic Scanning for Methylation (RLGS-M) and identified several loci/spots that appeared to be methylated frequently in liver tumors. One of these spots, B236, identified a locus on chromosome 12 (D12Ncvs7) syntenic with human 14q12-q21 that is frequently lost in certain human cancers. Shotgun sequencing of a bacterial artificial chro mosome clone containing this spot/locus was performed to identify genes within this region. The Genescan program predicted an open reading frame of a novel, intron-less gene adjacent to the B236 spot that encodes a putative 493-amino acid protein containing the SNAG repressor motif in the NH2-terminal region and five C2H2-type zinc finger motifs in the COOH-terminal half. This putative gene, methylated in liver tumor (mlt 1), is a novel member of the SNAG transcriptional repressor family with 43% amino acid identity to insulinoma-associated protein 1. An open reading frame encoding a protein quite similar to mouse mlt 1 (56% amino acid identity) was located in the syntenic region of the human genome, indi cating that mlt 1 is evolutionarily conserved in human. Northern blot analysis revealed that mlt 1 is normally expressed in brain, spleen, stom ach, and liver. However, mlt 1 expression was silenced in the liver tumors of MT-D2 mice. The putative promoter region of mlt 1 is unmethylated in normal tissues but methylated in all liver tumors from 11 MT-D2 mice We also found that mlt 1 was methylated and not expressed in N18TG-22 cells, a mouse neuroblastoma cell line. Treatment of N18TG-2 cells with a demethylating agent, 5-aza-deoxycytidine, resulted in an expression of mlt 1, indicating that the repression of mlt 1 is attributable to methylation Thus, mlt 1 is a novel target gene that is silenced by methylation during liver tumorigenesis initiated by SV40 T antigen.

The bio-industrial application of microalgae has gained much attention in recent years. One of the challenges in this field is to lower the cultivation and harvest costs and to achieve the steady productivity. To address this, new systems have been proposed, in which the products are extracted without killing the algal cells. These non-destructive extraction systems are called “milking.” Some of the milking systems reported so far are continuous processes where culturing and milking occur simultaneously, and the others are a periodic process where cells are cyclically cultured and milked. These systems are based on the organic solvent extraction of non-polar products, such as lipids, terpenes, and carotenoids. However, a special facility required for handling organic solvents increases the costs and solvents lost during milking need to be recouped. In this study, we examined a solvent-free method, based on mechanical milking using a shearing disperser (glass homogenizer). We cultured the N2-fixing filamentous cyanobacteria, Tolypothrix sp. PCC7601 in non-sterile agricultural water and performed long-term (87 days) milking cycles to harvest the extracellular carbohydrates and phycobiliproteins. As a result, the productivity of extracellular carbohydrates and the cell densities remained constant throughout the milking cycle, yielding 90–140 mg/L of extracellular carbohydrates every 3 weeks. Our results demonstrated that mechanical milking is a practical and effective method that can be used to harvest products from algae steadily.

Considering that agglomerates are subject to breakdown into individual flocs under shear, a theoretical model is developed to describe the shear viscosity behavior of flocculated suspensions using the Robinson equation ηa/μ0 = 1 + kφ/(1 − φ/φmax). The rheological properties of coal—oil mixtures (COM) have been investigated using a concentric cylinder rotational viscometer in the coal volume fraction range of 0.268–0.375 at 55–85 °C. The model successfully accounts for the effects of solid concentration and shear rate on rheological behavior of flocculated suspensions of irregularly shaped particles. The experimental results show that the agglomerates are densely packed with flocs, and are broken into small fragments under shear. The effect of temperature on the rheological behavior is also examined. By applying the shear rate—temperature superposition method, it was found that the bulkiness of agglomerates increases above 75 °C, leading to a change in the rheological properties of COMs.

We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length normalized cDNA libraries, of which the average residual polyA tail was 4 bases or shorter in most of the clones analyzed Because of the removal of homopolymeric stretches, libraries prepared with this method can be used for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.

This chapter proposes a new approach to recover 3-D shape from a Scanning Electron Microscope (SEM) image. When an SEM image is used to recover 3-D shape, one can apply the algorithm based on the solving the Eikonal equation with Fast Marching Method (FMM). However, when the oblique light source image is observed, the correct shape cannot be obtained by the usual one-pass FMM. The approach proposes a method to modify the original SEM image with intensity modification by introducing a Neural Network (NN). Correct 3-D shape could be obtained using FMM and NN learning without iterations. The proposed approach is demonstrated through computer simulation and validate through experiment.

長鎖状アルキレンオキシドとして1,2-エポキシオクタン,1,2-エポキシデカンおよび1,2-エポキシドデカンを合成して, これらのオキシドと水酸基を含む種々の化合物とを反応させた。水酸基を含む化合物として水, エタノール, エチレングリコール, グリセリン, D - ソルビットおよびフェノールを用い, 酸触媒およびアルカリ触媒下の条件で行なった。酸触媒の場合には,反応物の種類によってエポキシ基の開環の方向が異る場合があるのに対し,アルカリ触媒の場合には,常に開環は二級水酸基を生じる方向に起ることが推察された。このほか,水およびフェノールとの生成物から酸化エチレン付加物を合成し,その界面活性を従来の一価アルコールの酸化エチレン付加物と比較検討した結果,一価アルコールの酸化エチレン付加物にくらべて,表面張力および起泡力はほとんど変らないが流動点が低く,消泡性にすぐれていることがみとめられた。

Reactions and utilizations of higher alkylene oxides were investigated. The used oxides were 1, 2-epoxyoctane, 1, 2-epoxydecane and 1, 2-epoxydodecane synthesized from corresponding α-olefins by using peracetic acid. These oxides reacted easily with some organic acids, hydroxy compounds and amino compounds as compounds having active hydrogen. Higher alkylene oxides reacted also with benzene in the presence of aluminium chloride and could be polymerized in the presence of the same catalysts that had been used for the polymerizations of lower alkylene oxides. Furthermore, utilizations of the products in the reactions of oxides with some compounds having active hydrogen were investigated.

高齢化社会を迎え，認知症などの脳疾患に対する治療が必要とされている．筋肉の疲労や障害に対して手技マッサージが血行を促進するように，今回我々は浸透度が大きい長波超音波（30 kHz，2 mW/cm2以下）の経頭蓋微弱超音波振動刺激装置を用いて，健常者の頭蓋前頭部あるいは後頭部から照射することで，脳の血流がどのように変化するかを検討した． 方法は，本装置を前頭部あるいは後頭部に装着して，振動させる前後での脳内血流変化を測定した．すなわち，健常成人において，まず前頭部のみの照射でXeガスＸ線CT画像による測定を行い，次に，前頭部と後頭部の照射によるSPECT画像の変化を測定した．その結果，本装置の発する数ミリワットの微弱超音波振動により，脳血流量が増加することが示された．

アルドール縮合による2-エチルヘキサノールの工業的合成における蒸留残分の有効利用を目的として,その成分検索を行なった。蒸留残分は遊離酸6%(重量),遊離アルコール45%,エステル17%および残部からなるとみなされ,遊離酸およびエステルはそれぞれ主として2-エチルヘキソイン酸および2-エチルヘキソイン酸2-エチルヘキシルからなり,遊離アルコールは2-エチルヘキシル,デシル,ドデシルアルコールのほか,テトラデカンジオールも含むと考えられた。さらに2-エチルヘキソイン酸のエステルが,直鎖の脂肪酸からなるエステルより著しく加水分解をうけにくい事実を見出し,その原因を究明した。

Abstract The aim of this study was to examine the impact of the blue light from the visual display terminal (VDT) on visual search tasks. We assessed the quantity of the blue light from medical imaging diagnoses (medical‐grade) liquid‐crystal display (LCD) and general (personal computer‐grade) LCD for the VDT work, both equipped with the blue light reduction function. Then, we measured the visibility of display by evaluating the correct answer rate and reaction time of visual search tasks. The quantity of the blue light was proportional to the luminance of the display. When the blue light reduction function was used during the VDT work, the blue light at the peak wavelength was reduced by about 56%. Though the luminance of the display reduced, the visibility of the display was not decreased. These findings suggest that the blue light reduction on the LCD could work efficiently in the VDT work, maintaining the visibility of the display.

長鎖状アルキレンオキシドのひとつである1,2-エポキシドデカンと種々のアミノ化合物との反応について研究を行なった。アンモ二アのほかアミノ化合物として,第一アミン,第ニアミン,酸アミド,モノ-およびジエタノールアミンおよびピリジニウム塩を用いた。アンモニアおよび低級脂肪族第一アミンとの反応では,1分子のエポキシド付加物のほかに2分子のエポキシド付加物(それぞれ第ニアミンおよび第三アミンとなる)がえられ,エタノールアミンとの反応では,アミノ基が水酸基より優先的にエポキシドと反応するものと考えられ,たまた臭化ピリジニウム塩との反応では,中間体としてブロムヒドリソが生成することをみとめた。エポキシ基の開環は,二級水酸基を生じる方向に起こるものとおもわれた。

Combative sports are characterized by skills in rapidly switching from offense to defense, and it is considered difficult to acquire such skills. The purpose of this study was to design a combative sport (Kendo) class unit and examine its effectiveness. Two teaching materials involving a form of exaggeration (game modification) were developed to aid better understanding. The participants were 39 7th grade students (19 boys and 20 girls), one student teacher, and another teacher who had more than 20 years teaching experience. Both teachers were Kendo rank holders. Eleven physical education classes were conducted as the experimental Kendo teaching unit. Qualitative and quantitative data were gathered. The qualitative data source was a journal written by the teachers, sentences written by the students on their learning sheets, comments made by students at interviews about their thoughts during Kendo training, and those made by teacher's colleagues during class observation. The quantitative data source was a motivation questionnaire for formative evaluation of physical education classes developed by Takahashi et al. (1994), and the students' game performance. The motivation questionnaire and student game performance were analyzed by descriptive statistics whereas qualitative data were analyzed by the method of SCAT (Otani, 2007). The results of this study indicated that the student's skill learning motivation and skill acquisition tended to be high, when analyzed through the students' game performance through exaggeration games and mini-games. It suggested that the approach employed in this unit was successful, and that the exaggeration games applied in the unit were effective for concurrent skill acquisition. We consider that modification of games for combative sport training should be developed more actively.

Toshitatsu Suzuki , Kazuhiro Shibata , Somboon Theerawisitpong , Yasuo Watanabe 1 1 Department of Electrical and Electronics Engineering, Nippon Institute of Technology 4-1 Gakuendai, Miyashiro-machi, Minamisaitama-gun, Saitama-ken, 345-8501, Japan Tel: +81-480-34-4111, Fax: +81-33-7680 t_szki@m.ieice.org, somboon@wata-lab.com, watanabe@nit.ac.jp 2 Shinko Industries Inc. 3-28-12 Daita, Setagaya-ku, Tokyo, 155-0033, Japan Tel: +81-3-3413-9645, Fax: +81-3-3413-9610 s-k-shinkou1974@hkg.odn.ne.jp

炭素数8,10および12の高級α-オレフィンのエポキシドと種々の有機酸との反応から,14種の1,2-アルカンジオールのジエステルを合成し,それらの性状を調べ,潤滑油および可塑剤としての性能試験を行なった。潤滑油としては,芳香族酸エステルの性状が著しく劣るのに対し,脂肪族酸エステルは全般に良好な性状を示し,可塑剤としては,逆に芳香族酸エステルの方が優秀な性能をもつことがみとめられた。このほか,潤滑油試験の結果から,分子構造と主として粘度性状ならびに流動点との関係について検討した。

Transcriptional Sequencing (TS) is a novel DNA sequencing method based on the chain termination method using RNA Polymerase (RNAP). Transcriptional sequencing differs considerably from conventional cycle sequencing methods using DNA Polymerases (DNAPs) with 2′-deoxyribonucleotide triphosphates (2′-dNTPs) and 2′, 3′-dideoxyribonucleotide triphosphates (2′,3′-ddNTPs). The TS method employs a modified T7, T3, or SP6 RNAP with ribonucleotide triphosphates (rNTPs) and 3′-deoxyribonucleotide triphosphates (3′-dNTPs) as substrates and terminators, respectively, and the T7, T3, or SP6 promoter sequence is used as initiation site instead of the primer sequence. The characteristics of RNAP allow TS to be conducted under conditions that are substantially different from conventional cycle sequencing using DNAP. In TS, the reaction is performed isothermally (at 37°C) and is stable with high processivity. Transcriptional sequencing can be applied to various amounts of double-stranded DNA template because RNAP produces a large number of transcribed RNA molecules and the quantity of product is limited by the quantity of substrate rather than that of the template. Furthermore, in direct sequencing using TS, PCR products do not need to be purified before the sequencing reaction because RNAP requires neither 2′-dNTPs nor single-stranded PCR primers, but polymerization is primed using specific double-stranded promoters and rNTPs. This characteristic feature ensures that even highly GC-rich DNA templates can be sequenced effectively. Transcriptional sequencing is also applicable to MALDI-TOF-MS DNA sequencing owing to the stability of the RNA polymer against fragmentation caused by laser ablation. Thus, TS differs considerably from conventional cycle sequencing methods using thermostable DNA polymerases, but should complement such methods in numerous practical applications.

In cDNA project which is to collect various cDNAs and determine the full-length sequences, theselectionofcloneisthemostimportantsteptoreducetime,laborandcost. cDNAlibrariesconsistsof clones derived from same loci, alternatively spliced, or some terminal sequence variants. For theselectionofclones,wehavedevelopedthesystemthatcategorizesclonesbasedonbothendsequenceswithtwokindsofcriteria. Oneisfortheselectionofallkindsoftranscripts,andtheotherisoftheclonederivedfromeachlocus.

Our Mouse Encyclopedia Project, which performs exhaustive collection and sequencing of all mRNA species expressed in mouse, successfully collected more than 70,000 different kind of cDNA clones classifiable by 3’-end sequence tags. By Using those clones, we are completing sequence of the isolated cDNA clones in concurrent manner, which piled up to over 10,000 sequences. This number tells that our project reached to the new realm where other genome project could not have reached in terms of the total number of genes sequenced. In contract to the genomic sequencing, our strategy choosing cDNA as the sequencing material brought us big advantages that the amount of sequencing required to obtain all gene information requires much less effort in terms of the number sequence runs, and each gene information are ready for application, upon completion of individual sequence with out further artificial processing of data, i.e. prediction of gene region and exon structure. By taking this advantage, we have started several projects such as analyses of expression profiles and proteinprotein interaction. In the current study, we report results of one of such application, produced by bioinformatic approaches.